Insulin-like growth factor binding proteins (hereinafter, referred to as “IGFBP”) are a group of molecules that were discovered due to the fact that the insulin-like growth factor (hereinafter, referred to as “IGF”) exists in the body fluid as a macromolecular complex. 10 types of these molecules, IGFBP-1 to 10, have been reported to exist until now, and are known to constitute a superfamily (Endocr. Rev., 18, 801 (1997); Prog. Growth Factor Res., 3, 243 (1991); Mol. Reprod. Dev., 35, 368 (1993); Proc. Natl. Acad. Sci. USA, 94, 12981 (1997)).
The existence of direct action by binding to integrin and the like has been also suggested as the function of IGFBP. However, the main action is predicted to be exhibited by the binding to IGF and insulin to regulate their activity, distribution, metabolism, and the like (Endocr. Rev., 18, 801 (1997); Bio Science Terminology Library Cytokines and Growth Factors, revised edition p14–17 (1988)). Specifically, the IGFBP is expected to exhibit its function by suppressing the transport into blood, extravasation and degradation of IGF, by regulating receptor binding, and the like.
6 types of molecules, IGFBP-1 to 6, among the IGFBP superfamily have structural similarlity and bind with higher affinity to IGF than to insulin. Therefore, they are classified into a subfamily as high-IGF-affinity IGFBPs (Mol. Endocrinol., 2, 404 (1988); EMBO J., 8, 2497 (1989); Mol. Endocrinol., 2, 1176 (1989); Mol. Endocrinol., 4, 1806 (1990); Biochem. Biophys. Res. Commun., 176, 219 (1991); J. Biol. Chem., 266, 9043 (1991); J. Biol. Chem., 266, 10646 (1991)).
In humans, the amino acid sequence homology among the molecules belonging to the high-IGF-affinity IGFBP subfamily is 49 to 60%. Furthermore, 18 cysteine residues are conserved in five of the IGFBPs, excluding IGFBP-6, and three of these residues to the N-terminal side form a homologous sequence represented by Gly-Cys-Gly-Cys-Cys-X-X-Cys (SEQ ID NO:26) (X represents an arbitrary amino acid; this homologous sequence is called insulin-like growth factor binding motif (hereinafter, refered to as “IGFBP motif”)) and has been revealed to be involved in IGF binding (Prog. Growth Factor Res., 3, 243 (1991)).
IGFBP-1 and 3 are known to both suppress and enhance the action of IGF. Whereas IGFBP-2, 4, and 6 are inhibitory IGF binding proteins and IGFBP-5 as a promoting IGF binding protein. There are two types of IGFs, namely, (1) IGF-I: produced in a growth hormone dependent manner in liver, bone tissue, and the like, and functions as a growth factor that promotes physical growth; and (2) IGF-II: expressed in large amounts in central nervous system, bone tissue, and the like, and is presumed to play an important role in growth, mainly during the embryonal period. IGFBP-1, 3, and 4 indicate a binding activity similar toward IGF-1 and IGF-II, while IGFBP-2, 5, and 6 indicate a strong binding activity mainly toward IGF-II.
The tissue distribution of high-IGF-affinity IGFBP subfamily molecules differ depending on the molecules. IGFBP-1 is known to mainly exist in amniotic fluid and fetal serum; IGFBP-2 mainly in fetal liver and adult brain; IGFBP-3 mainly in liver and serum; IGFBP-4 mainly in renal glomeruli, skin, and intestinal epithelium; IGFBP-5 mainly in intestinal epithelium and bone; and IGFBP-6 mainly in skin and heart.
The following findings have been reported regarding the relationship between IGFBPs and pathology of diseases.
The fluctuations of IGF and IGFBP expressions are directly associated with the pathophysiology of dwarfism and acromegaly patients. On the other hand, although failure to thrive are often seen among patients of infantile chronic renal failure, their growth hormone and IGF expression levels are normal, and most of the cause is the functional disorder of IGF due to the increase of IGFBP-2 and/or IGFBP-3 (Miner. Electrolyte Mrtab., 18, 320 (1992)). IGFBP-4 and 5 have important functions in bone metabolism. It has been reported that the expression level of IGFBP-5 decreases in osteoporosis, and the expression of IGFBP-4 elevates in women fracture patients of old age accompanied with the elevation of parathyroid hormone. Furthermore, although enhancement of paracrine effect of IGF-I is observed in compensatory hypertrophy after nephrectomy and small intestine resection, the mRNA level of IGF-I is invariant, and free IGF-I increases due to diminished IGFBP-3 expression (J. Fuller, Baillieres Clin. Endocrinol. Metab., 8, 165 (1994)). Moreover, the expression of IGFBP-1 is decreased in malignant tumors of endometrium, compared to benign tumors (Growth Regul., 3, 74, (1993)).
On the other hand, four molecules in the IGFBP superfamily, IGFBP-7 to 10, are structurally similar and are considered to share a common characteristic of having a low affinity to IGFs. Therefore, they are classified into a separate subfamily as low-IGF-affinity IGFBPs (Proc. Natl. Acad. Sci. USA, 94, 12981 (1997); Cancer Res., 59, 2787 (1999)).
Regarding IGFBP-7, various physiological activities have been reported. Particularly, many reports have been made on the relationship with cancer and its pathology.
While the expression of IGFBP-7 is elevated in aged human epithelial cells (J. Clin. Endocrinol. Metab., 4, 715 (1993)), its expression is diminished in carcinoma cell lines. Therefore, IGFBP-7 is thought to function as a gene involved in cancer suppression activity (Proc. Natl. Acad. Sci. USA, 92, 4472 (1995)). The gene locus of IGFBP-7 on the human chromosome is 4q12, and its expression is known to enhance by the treatment of human epithelial cells with retinoic acid (Proc. Natl. Acad. Sci. USA, 92, 4472 (1995)).
In breast cancer tissues, LOH (loss of heterozygosity) at chromosome 4q12 to 13 was observed at a frequency of approximately 50%, and the expression of IGFBP-7 has been confirmed to be decreased (Oncogene, 16, 2459 (1998)). The IGFBP-7 expression is also decreased at the mRNA level in prostate cancer tissues, and no expression is detected particularly in cell lines derived from malignant prostate cancer (J. Clin. Endocrinol. Metab., 83, 4355 (1998)).
Moreover, when IGFBP-7 is forcedly expressed in prostate cancer cell line in which IGFBP-7 expression is decreased, extension of cell division time, decrease of colony forming ability in soft agar medium, decrease of tumor forming ability in nude mouse transplantation, and elevation of apoptosis induction rate by drug treatment have been observed, which suggests a relationship between the IGFBP-7 expression and degree of malignancy of prostate cancer (Cancer Res., 59, 2370 (1999)).
Concentrations of IGFBP-7 and IGFBP-3 in the cerebrospinal fluid have been reported to rise in leukemia patients, and their relationship to the pathology of leukemia is receiving attention (J. Clin. Endocrinol. Metab., 84, 1283 (1999)).
Due to the enhanced expression of IGFBP-7 in large intestinal cancer tissues and cell lines, the relationship of IGFBP-7 with the pathology of colon cancer is receiving attention (J. Gastroenterology, 33, 213 (1998)).
IGFBP-7 expression is decreased in large uterine leiomyoma sites, and the expression of IGFBP-7 is reported to be elevated in patients who have received gonadotropin-releasing hormone therapy (J. Reprod. Immunol., 43, 53 (2000)).
The 5′ upstream region of IGFBP-7 gene is methylated and its expression level is decreased in mouse liver cancer cells that have been induced with SV40T antigen. Therefore, a mechanism involving methylation of the gene has been proposed for the regulation of the IGFBP-7 expression associated with canceration (Biochem. Biophys. Res. Commun., 267, 109 (2000)).
The relation between IGFBP-7 and diabetes has also been reported.
A factor (PGI2-stimulating factor, hereinafter, referred to as “PSF”) promoting the production of prostacyclin PGI2 by acting on vascular endothelial cells was shown to be identical to IGFBP-7 (Biochem. J., 303, 591, (1994)). The expression of this factor, which is expressed in vascular endothelial cells and smooth muscle cells (Thromb Haemost., 74, 1407 (1995)), is reported to be decreased in kidneys and angiopathy sites of a type I diabetes model established by streptozotocin administration (Diabetes, 45, S111 (1996); J. Diabetes & its Complications, 12, 252 (1998)). Furthermore, decreased expression of IGFBP-7 at the protein level has also been observed in coronary artery smooth muscle cells of type II diabetes patient (Diabetes, 46, 1627 (1997)). Moreover, the expression level of IGFBP-7 has been confirmed to decrease at the mRNA and protein levels by culturing smooth muscle cells derived from bovine arteries in high-glucose medium (Diabetes, 46, 1627 (1997); Diabetologia, 41, 134 (1998)).
The expression of IGFBP-7 in osteoblasts rises by TGF-β (transforming growth factor-β), parathyroid hormone (PTH), and prostaglandin E2 (PGE2). Therefore, IGFBP-7 appears to have physiological effect on osteoblasts (Endocrinology, 140, 1998 (1999)). Furthermore, treatment of osteoblasts with glucocorticoids is reported to suppress the expression of IGF-I, while enhancing the expression of IGFBP-7 (Endocrinology, 140, 228 (1999)).
Furthermore, IGFBP-7 is indicated to have an effect on the differentiation into skeletal muscles by suppressing the differentiation-promoting action of IGF (Exp. Cell Res., 237, 192 (1997); Endocrinology, 141, 100 (2000)).
Since IGFBP-7 was the same molecule as the factor PSF that promotes prostacyclin PGI2 production, a part of the physiological function of IGFBP-7 is considered to perform as the effector molecule for PGI2.
PGI2, a type of prostaglandin, has strong platelet aggregation inhibitory effect and vasorelaxing effect, and is known to function antagonistically with TXA2 that has the opposite effect to maintain the homeostasis within a living body (Br. J. Pharmac., 76, 3 (1982)). Imbalance in TXA2 and PGI2 production, especially decrease in PGI2 production causes development of angiopathy in thrombosis and arteriosclerosis (Br. J. Pharmac., 76, 3 (1982)). Regarding the development and progression of diabetic angiopathy, in addition to the enhancement of platelet-derived TXA2 production (Thromb. Res., 19, 211 (1980); J. Lab. Clin. Med., 97, 87 (1981)), decrease of blood vessel-derived PGI2 production has been confirmed to enhance platelet aggregation in diabetes patients and in laboratory animals with diabetes (Lancet, 1, 325 (1979); Lancet, 2, 1365 (1979); N. Engl. J. Med., 300, 366 (1979); Life Sci., 23, 351 (1978)).
Furthremore, PSF is a factor that exists in the blood stream and is reported to stimulate the production of PGI2 in vascular wall (Nature, 271, 549 (1978)). Moreover, the level of this factor in blood is reported to be decreased in hemolytic uremic syndrome (Lancet, 2, 871 (1978)), thrombotic thrombocytopenic purpa (Lancet, 2, 748 (1979)), sickle cell anemia (Br. J. Haematol., 48, 545 (1981)), acute myocardial infarction (Coronary, 2, 49 (1985)), diabetic angiopathy (Metabolism, 38, 837 (1989); Haemostasis, 16, 447 (1986); Diab. Res. Clin. Pract., 3, 243 (1987)), and arteriosclerotic diseases.
More specifically, IGFBP-7 has been demonstrated to exert platelet aggregation inhibitory effect, smooth muscle relaxation effect, and gastric acid secretion inhibitory effect by elevating the PGI2 concentration in blood via the promotion of PGI2 production of vascular endothelial cells.
As mentioned above, factors belonging to the IGFBP superfamily have been revealed to be involved in various biological phenomena including regulation of IGF and insulin function, pregnancy, compensatory effect in exhaustive diseases, bone metabolism, differentiation of skeletal muscle cells, promotion of PGI2 production in vascular endothelial cells, PGI2-mediated inhibition of platelet aggregation, vascular smooth muscle relaxation, bronchial smooth muscle relaxation, and inhibition of gastric acid secretion. Furthermore, they are shown to be associated with diseases, such as dwarfism, acromegaly, infantile chronic renal failure, osteoporosis, breast cancer, prostate cancer, acute leukemia, large intestine cancer, uterine leiomyoma, liver cancer, type I diabetes, type II diabetes, thrombosis, arteriosclerosis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpa, sickle cell anemia, acute myocardial infarction, and diabetic angiopathy.
Therefore, proteins having the activity of IGFBP that belong to the IGFBP superfamily, genes encoding the proteins, antisense DNAs, and antibodies recognizing the proteins are considered to serve as medicaments for detecting, treating, or preventing diseases accompanying abnormal cell growth, diseases accompanying angiopathy, diseases accompanying abnormal bone metabolism, diseases accompanying disorders of IGF and growth hormone action, diseases accompanying abnormal differentiation or growth of smooth muscle cells, diseases accompanying abnormal differentiation or growth of skeletal muscle cells, diseases accompanying abnormal gastric acid secretion, or inflammatory diseases accompanying abnormal lymphocyte invasion. Thus, great attention, as useful targets for development of new drugs, has been paid on factors belonging to the IGFBP superfamily.
Furthermore, the possibility on the existence of novel factors belonging to the IGFBP superfamily was indicated. By obtaining a novel IGFBP gene, the function of this IGFBP can be estimated by comparing the amino acid sequence of the novel IGFBP with that of known IGFBPs, or by studying the expression distribution of transcription products of the IGFBP gene, to finally provide useful information for drug development. In addition, when a novel IGFBP gene is obtained, substances that suppress the expression or function of the IGFBP can be screened. Compounds obtained by this screening procedure are expected to serve as useful drugs.